2. E.coli K-12 MG1655 pathway 調節 磷酸果糖激酶 烯醇酶 缺少 pyruvate decarboxylase
alcohol dehydrogenase

4. Anaerobic fermentation pathway of non-growing yeast, with glucose as the sole carbon source
。Solid arrows : reaction steps
。 Dashed arrows : activation
。 Dotted arrows : inhibition
。 regulatory loops # (+,-) : 7
Vassily Hatzimanikatis
accepted 30 July 1997

5. How to maximize specific ethanol production?
Which of the existent regulatory loops should be inactivated?
What associated changes should be made in enzyme expression levels?
Three enzymes must be
over-expressed
Two activation loops should be retained
‧This method will maximize ethanol production, giving an increase of 100% relative to
the reference state.

6. E.coli & Yeast regulatory superstructure
‧Dashed arrows : yeast
‧ Dotted arrows : E. coli
‧ Dashed-dotted : yeast & E.coli

7. The optimal regulatory structure
This method will maximize ethanol production, giving an increase of 114% relative to
the reference state.

8. 17%
11%
10%
Specific glucose uptake(B) and ethanol production rate(A) at 0.5% initial glucose
concentration of anaerobic, resting E. coli KO20 strains with the plasmid pTrc99A
only (control), overexpression pfkF from E. coli(PYKec), and overexpression pyk
From B. stearothermohilus(PYKbs). Cells were harvested from anaerobic mid-exponential growth
phase cultures. The indicated values represent the mean value of at least
two independent experiments, and error bars indicate the standard deviation.

9. Specific glucose uptake (B) and ethanol production rate (A) at 2% initial glucose concentration of anaerobic, resting E. coli KO20 strains with the plasmid pTrc99A only (control), overexpressing pfkF from E. coli (PYKec), and overexpressing pyk from B. stearothermophilus (PYKbs).Different shading indicates different time points after the start of the experiment: 3 h (light grey), 5 h (grey), and 7 h (dark grey). Cells were harvested from the late exponential growth phase of aerobically grown cultures. The indicated values represent the mean value of at least two independent experiments, and error bars indicate the standard deviation.

14. History Records system biology tools we try to use
Many tools for pathway analysis
Gepsi, Copsi, and FluxAnalysis
Advantage : using kinetic parameters with accurate qualitative analysis.
Disadvantage : without kinetic parameters knowledge, must mine information from literature or obtain from experiments

15. GNU Linear Programming Kit
GNU Homepage
GNU Free Software Directory
Environment
Unix / Win32 (4.1 version)

16. Linear Programming Reaction File Compound file Object file
Perl program
Manual operation
Execute glpk instruction
Result
file

17. Reaction File Context context
R C C00011 <=> C C00022
R C C00022 <=> C C00074
R C C C00010 <=> C C C00024
R C00631 <=> C C00001
R C C C00001 <=> C C C00080
R C C00003 <=> C C C00080
R C C C00003 <=> C C C00080
R C05378 <=> C C00118
R C C00197 <=> C C00236
R C00631 <=> C00197
R C C00579 <=> C C01136
R C C00031 <=> C C00668
R C00668 <=> C05345
R C C00248 <=> C C00068
R C C05345 <=> C C05378

18. Compound File Context Context We must mutually add
C00001: + R R00710 >= 0
C00002: - R R R R04779 >= 0
C00003: - R R R01061 >= 0
C00004: + R R R01061 >= 0
C00008: + R R R04779 >= 0
C00010: - R R02569 >= 0
C00022: + R R00200 >= 0
C00024: + R R02569 >= 0
C00033: - R R00710 >= 0
C00068: + R R03270 >= 0
C00074: + R R00658 >= 0
C00080: + R R R01061 >= 0
C00084: - R R00754 >= 0
C00118: - R R01070 >= 0
C00197: - R R01518 >= 0
C00236: + R R01512 >= 0
C00631: - R R01518 >= 0
C00668: + R R02740 >= 0
C01136: + R R03270 >= 0
C05125: - R R03270 >= 0
C05345: + R R04779 >= 0
C05378: - R R04779 >= 0
C00469: - R00754 >= 0
C00000: + R02738 = 100
We must mutually add
ethanol compound constraint and input constraint into compound file, because It has only one reaction

19. Reaction & Compound Pathway

20. Reaction & Compound Pathway (modified)

21. Reference http://biocyc.org/ECOLI/ (EcoCyc)
(KEGG)
David R. Shonnard, “Applications of Molecular Biotechnology Ethanol Production from Cellulosic Biomass”,CM4710 Biochemical Processes, November 28, 2003.

22. 謝謝各位